Stress protein epitopes

ABSTRACT

A functional epitope which is purified from human HSP 90 or which is synthesised to correspond to such a purified epitope, which is, if purified, unchanged or changed by substitution of selected amino acids and if synthesised is identical to a purified epitope or differs from a purified epitope by substitution of selected amino acids, and which cross-reacts with an antibody raised against a stress protein.

This invention concerns stress protein epitopes intended inter alia foruse in diagnosis and treatment of disease states in which stressproteins are produced.

Environmental stress can induce an increase in the rate of synthesis ofso-called heat shock, or stress, proteins in both procaryotic andeucaryotic cells (see for example Schlesinger et al (eds) in: Heat Shockfrom Bacteria to Man, Cold Spring Harbor, N.Y., (1972)). Although thefunction of the stress proteins has yet to be finally resolved, somehave been reported to participate in assembly and structuralstabilisation of certain cellular and viral proteins, and their presenceat high concentration may have an additional stabilising effect duringexposure to adverse conditions.

Many pathogenic organisms have been shown to produce stress proteins(see Young et al, Proc. Natl. Acad. Sci. USA, 85, 4267-4270 (1988)). Theproteins are thought to be produced in response to the stress ofinfection to help protect the invading pathogen. Thus, for example, theability to produce stress proteins has been implicated in the survivalof bacterial pathogens within macrophages (Christmas et al, Cell, 41,753-762 (1985) and Morgan et al, Proc. Natl. Acad. Sci. USA, 83,8059-8063 (1986)).

Burnie et al, (GB 90307236.1, WO 92/01717), have found that stressproteins from both fungi and bacteria, for example, Candida albicans andCorynebacterium leikium, comprise an immunodominant conserved antigen.The carboxy end of the Candidal stress protein has been sequenced and anantibody raised against the epitope LKVIRKNIVKKMIE found to recogniseboth the 47 and 92 Kd Candidal stress proteins in sera from patientssuffering from systemic Candidal infection. In addition, the antibodiesalso recognised stress proteins in sera of patients suffering from otherfungal infection, for example, the 40 and 88/84 Kd Aspergillus stressproteins, as well as stress proteins in sera from patients sufferingfrom bacterial infection, for example, the 52 and 86 Kd Coryneformstress proteins. Other peptide sequences from the Candidal stressprotein were found to be immunogenic, for example, the epitopes LSREM,LKVIRK and STDEPAGESA reacted with sera from patients with systemiccandidiasis.

The entire human 89 kDa heat shock protein (HSP90) gene has beensequenced (Hickey et al, Mol. Cell. Biol., 9, 2615-2626, 1989) and itsamino acid sequence deduced and compared to that of heat shock proteinsof other species. Although it appears that the class of heat shockproteins is highly conserved among species, direct comparison andidentification of common functional sequences (i.e. epitopes) of theheat shock proteins have not been reported.

It is now found that, notwithstanding the efficacy of the earlierdescribed epitopes, routes to production, other than from the carboxysequence of the candidal HSP90, can give equal or potentially superiorresults when used in diagnosis or therapy.

According to the present invention there is provided a functionalepitope which is purified from human HSP 90 or which is synthesised tocorrespond to such a purified epitope, which is, if purified, unchangedor changed by substitution of selected amino acids and if synthesised isidentical to a purified epitope or differs from a purified epitope bysubstitution of selected amino acids, and which cross-reacts with anantibody raised against a stress protein.

The epitope may comprise the amino acid sequence XXXLXVIRKXIV, orXXILXVIXXXXX, wherein X is any amino acid, and may comprise, forexample, the amino acid sequence NKILKVIRKNIV.

The epitope may be selected from the amino acid sequences KIRY, NNLGTI,QFIGYPI, KKIK, SKEQV or Candidal equivalent sequence SIKAV, GLELPE orCandidal equivalent sequence FELEES, LDKK or Candidal equivalentsequence LGDQ, WTAN or Candidal equivalent sequence WSAN, NSTMGY orCandidal equivalent sequence TTMSSY, PIVET or Candidal equivalentsequence PIIKE, or KNDK or Candidal equivalent sequence AEDK.

The stress protein may comprise a malarial stress protein, a fungalstress protein or a bacterial stress protein.

The fungal stress protein may comprise a Candidal 92 and/or 47 KDaprotein or an Aspergillus 40, 51 and/or 88/84 KDa protein, or a stressprotein of Pneumocystis carnii.

The bacterial stress protein may comprise a Coryneform 86 and/or 52 KDaprotein or a Streptococcal stress protein.

The invention also comprises, in another aspect, a method of making afunctional epitope from human HSP 90 or by synthesis, comprising thestep of purification from human HSP 90 with or without substitution ofselected amino acids or of synthesis of an epitope which is identical toa purified epitope or which differs from a purified epitope bysubstitution of selected amino acids, the amino acid subsitution beingselected so that the epitope cross-reacts with an antibody raisedagainst a stress protein.

The epitope according to the invention is described as a functionalepitope and, as such, has a number of functional uses. In particular itmay be used in the diagnosis and treatment of a number of diseases as analternative and/or improvement to conventional diagnostic andtherapeutic methods. The present invention may, for example, be used inthe diagnosis and treatment of malaria. This is of topical importancebecause, as is well known, this disease is fast becoming resistant tocurrent drug treatments, and, as a consequence, is becoming moreprevalent throughout the world.

The functional epitope may form the basis of a diagnostic test formalaria, fungal infection, including Pneumocystis carnii or bacterialinfection, using an immunological test such as an enzyme-linkedimmunosorbant assay, a radioimmunoassay or a latex agglutination assay,essentially to determine whether antibodies specific for the epitope,and therefore the particular stress protein, are present in a hostorganism. The test may be generally formed by contacting body fluid fromthe host with an epitope and detecting any complexed material.

In another use, the epitope according to the invention may be employed,using conventional techniques, for screening to obtain activityinhibiting agents for use in the treatment of malaria, fungal andbacterial infections.

In a further use, the epitope according to the invention may be used togenerate antibodies, ie. for use as an immunogen, by standardtechniques, for example, by injecting any suitable host with the epitopeand the serum collected to yield the desired polyclonal anti-epitopeantibody after purification and/or concentration. Prior to injection ofthe host, the epitope may be formulated in a suitable vehicle to providea composition comprising an epitope together with one or morepharmaceutically acceptable excipients.

Alternatively, the antibodies may be monoclonal in origin and may ingeneral belong to any immunoglobulin class, for example, IgG and/or IgMand/or IgA. The antibody may be of animal, for example, mammalian originand may be of murine, rat or preferrably human origin, or may be amurine or rat humanised antibody.

For purification of any anti-epitope antibody, use may be made ofaffinity chromatography employing an immobilised epitope of theinvention as the affinity medium. Such anti-epitope antibodies may beused in both the diagnosis and treatment of fungal and bacterialinfections and malaria. As inhibitors of the action of a stress protein,the anti-epitope antibodies may be used either alone or in combinationwith other pharmaceutical agents, For example, other anti-fungal agentsor anti-malarial agents. In addition, such epitopes may be used toproduce other inhibitors of fungal or malarial stress proteins, forexample, ribosymes and anti-sense RNA will inhibit the translation ofstress protein mRNA.

A potential use of such anti-epitope antibodies is in the supportiveimmunotherapy of, for example, HIV positive patients. Such patients areprone to opportunistic infections due to their immune system beingcompromised. Examples of such opportunistic infections include Candida,Aspergillus and Pneumocystis carnii. Indeed, sera from HIV positivepatients have been shown to be antibody positive to HSP90 of all ofthese organisms. It is thus proposed to provide such patients withantibody which will recognise HSP90 of these, and other infectousorganisms, before these infections become establised and contribute tothe death of such patients.

A particularly useful antibody according to the invention is that whichrecognises the peptide XXXLXVIRKXIV, or XXILXVIXXXXX, wherein X is anyamino acid, for example, the peptide NKILKVIRKNIV, and an antibody whichrecognises one or more of the peptides KIRY, NNLGTI, QFIGYPI, KKIK,SKEQV or Candidal equivalent sequence SIKAV, GLELPE or Candidalequivalent sequence FELEES, LDKK or Candidal equivalent sequence LGDQ,WTAN or Candidal equivalent sequence WSAN, NSTMGY or Candidal equivalentsequence TTMSSY, PIVET or Candidal equivalent sequence PIIKE, or KNDK orCandidal equivalent sequence AEDK.

If desired, mixtures of antibodies may be used for diagnosis ortreatment, for example mixtures of two or more antibodies recognisingdifferent epitopes of the human stress protein (or Candidal equivalentsequence), and/or mixtures of antibodies of a different class, forexample, mixtures of IgG, IgM and IgA antibodies recognising the same ordifferent epitope(s) of a human stress protein (or Candidal equivalentsequence).

The following examples illustrate the invention.

EXAMPLE 1

Possible immunodominant epitopes of the human HSP 90 have beeninvestigated against sera from patients suffering from various types ofdiseases in an attempt to provide novel tools for both diagnosis andtreatment of disease.

Sera examined included that from patients suffering from systemiccandidiasis (47 kDa positive), invasive aspergillosis (88,40 kDapositive), allergic bronchopulmonary aspergillosis, aspergilloma,malaria, Streptococcus faecalis endocarditis, Corneybacterium ieikeiumendocarditis and the autoimmune disease systemic lupus erythematosis(SLE).

Epitope mapping of human HSP 90 was carried out against the derivedamino acid sequence described in Hickey et al 1989, Mol. Cell. Biol., 9,2615-2626.

Experimental Details

The 716 amino acid residues were synthesised on polyethylene pins as acomplete set of overlapping nonapeptides, in which peptide 1 consistedof residues 1-9, peptide 2 of residues 2-10, etc. Peptide synthesis wasperformed with Fmoc-protected amino acid esters. The polyethylene pinsthemselves were each coupled to Fmoc-β-alanine. After Fmoc deprotection,the first amino acid was coupled to each pin as dictated by the sequenceto be synthesized. Hydrobenzotriazole-mediated coupling reactions werecarried out overnight in a N,N-dimethylformamide solution of each sidechain protected, Fmoc amino acid. Peptides were synthesized bysuccessive cycles of Fmoc deprotection and addition of one amino acidper pin per day. After completion of the final coupling reaction, andremoval of the Fmoc protecting group, the terminal amino group wasacetylated in order to remove the unnatural charge of the N terminus ofthe peptide. Side chain protecting groups were removed by a mixture oftrifluoroacetic acid: phenol: ethanedithiol (95: 2.5: 2.5, v/w/v).

The peptides, still coupled to the surface of the pins, were testedagainst sera by enzyme immunoassay (EIA). Pins were precoated for 1 hr.,in microtitre plates containing 1% ovalbumin, 1% bovine serum albumin(BSA) in PBS-T (phosphate-buffered saline, 0.1% Tween 20). They werethen incubated at 4° C. in patient sera (1/200) washed four times withPBS-T and incubated for 1 h with horseradish-peroxidase conjugatedantiimmunoglobulin (1/1000; Sigma, Poole). After further washing, thepins were immersed for 30 min in ABTS (0.5 mg/mlamino-di-3-ethylbenzthiazoline-6-sulphonate in pH 4.0 citrate bufferwith 0.03% hydrogen peroxide) and A₄₀₅ measurements made in an EIA platereader. Pins were cleaned by sonication.

A reaction was considered to be specific if, over at least three wells,the OD was at least 2-fold above background. The large number of thepeptides synthesized effectively acted as negative controls in eachtest. The mean absorbance of these peptides was low and was used toestablish the background level (Geysen et al., 1987).

DETAILS OF PATIENT'S SERA EXAMINED

I. Disseminated Candidiasis

    ______________________________________                                        Number    History                                                             ______________________________________                                        1.        leukaemia, neutropenia, blood culture positive                                C. albicans.                                                        2.        post oesophagectomy, blood culture positive,                                  C. albicans.                                                        3.        HIV positive, drug abuser, Candidal                                           chorioretinitis.                                                    4.        Candidal peritonitis, chronic ambulatory                                      peritoneal dialysis patient, blood culture                                    positive C. albicans.                                               5.        Post duodenal resection, positive for                                         C. albicans in two sets of blood culture.                           6.        Pre (antibody negative), Post antibody                                        positive. Positive for C. alibicans in two sets                               of blood culture. Post cholecystectomy.                             7.        Post oesophagectomy, chest drain, blood culture                               positive C. albicans.                                               8.        Hepatosplenic candidiasis, yeast seen on biopsy.                    9.        Chronic ambulatory peritoneal dialysis patient,                               peritonitis, abdominal abscess, positive for                                  C. albicans in two sets of blood culture.                           ______________________________________                                    

Antibody profile

    ______________________________________                                        Case Number                                                                            Candida albicans                                                                            Aspergillus fumigatus                                  ______________________________________                                        1.       M 47, 40 KD   NIL                                                             G 47, 40 KD                                                          2.       M 47 KD       88, 84 KD trace                                                 G 47 KD       (M + G combined)                                       3.       M 47, 48 KD   NIL                                                             G NIL                                                                4.       M 47 KD       NIL                                                             G 47 KD                                                              5.       M 47 KD       NIL                                                             G 47 KD                                                              6.       Pre NIL       NIL                                                             Post M 40, 47 KD,                                                             Post G 40, 47 KD                                                     7.       M 47 KD       NIL                                                             G 47 KD                                                              8.       11/12/91 NIL  NIL                                                             17/1/92 M + G                                                                 47 KD (positive when                                                          combined)                                                            9.       M 47 KD       88/84 KD trace                                                  G 47 KD       (M + G combined)                                       ______________________________________                                    

Comment

C.albicans HSP 90 has bands at 92, 47, 40 KD (as disclosed in GB 2 034504).

A.fumigatus HSP 90 has bands at 88/84, 51 and 40 KD.

HIV positive patients sera are antibody positive for Candida HSP90,Pneumocystis carnii HSP90 and Aspergillus. Thus, it is proposed to usethe antibody in maintenance therapy of such patients, since loss ofantibody in these patients may lead to opportunistic infections, such asPneumocystis carnii and other organisms which produce antigen i.e.HSP90.

II. Invasive aspergillosis

    ______________________________________                                        Case Number                                                                              History                                                            ______________________________________                                        10.        Fatal, invasive aspergiliosis, acute leukaemia                     11.        Fatal, invasive aspergiliosis, acute leukaemia                     12.        Fatal, invasive aspergiliosis, acute leukaemia                     13.        Survivor, invasive aspergiliosis, acute leukaemia                  14.        Survivor, invasive aspergiliosis, acute leukaemia                  15.        Survivor, invasive aspergiliosis, acute leukaemia                  ______________________________________                                    

Antibody Profile

    ______________________________________                                        Case Number Candida albicans                                                                          Aspergillus fumigatus                                 ______________________________________                                        10          NIL         NIL                                                   11          NIL         NIL                                                   12          NIL         M 88,84 KD,                                                                   G 88,84 KD                                            13          NIL         M 88,84 KD,                                                                   G 88,84 KD                                            14          NIL         M 88,84 KD,                                                                   G 88,84 KD                                            15          NIL         M 88,84 KD,                                                                   G 88,84 KD                                            ______________________________________                                    

III. Allergic bronchopulmonary aspergillosis

    ______________________________________                                        Case Number C. albicans Aspergillus fumigatus                                 ______________________________________                                        16.         NIL         M 88,84 KD,                                                                   G 88,84 KD                                            17.         NIL         M 88,84 KD,                                                                   G 88,84 KD                                            ______________________________________                                    

IV. Aspergilloma

    ______________________________________                                        Case Number C. albicans Aspergillus fumigatus                                 ______________________________________                                        18.         NIL         M 88,84 KD,                                                                   G 88,84 KD                                            ______________________________________                                    

V. Streptococcus faecalis endocarditis

    ______________________________________                                        19.      C. albicans        M 47 KD                                                                       G 47 KD                                                    A. fumigatus       88, 51 KD                                         ______________________________________                                    

The monoclonal antibody described in GB 2 034 504 crossreacts with aS.faecalis immunodominant band at 90 KD as did the rabbit serum raisedagainst this peptide (LKVIRKNIVKKMIE-cys-KLH).

Previous work (Burnie et al, 1985) identified this antigenic band asimmunodominant in S.faecalis endocarditis, suggesting that it may formthe basis of a test for culture negative endocarditis.

VI. Malaria

    ______________________________________                                               C. albicans      A. fumigatus                                          ______________________________________                                        20.      M 92 KD                                                                       G 47 KD            51, 40 KD                                         ______________________________________                                    

A further 5 cases of malaria have been immunoblotted against candidaland aspergillus extracts to show they have antibody which crossreactswith fungal HSP 90.

VII. Corynebacterium ieikeium endocarditis

    ______________________________________                                               C. albicans      A. fumigatus                                          ______________________________________                                        21.      M 47 KD                                                                       G 47 KD            NIL                                               ______________________________________                                    

VIII. Systemic lupus erythematosis (SLE)

    ______________________________________                                        C. albicans          A. fumigatus                                             ______________________________________                                        22.    G 47 KD           NIL                                                  23.    NIL               M 88, 84 KD, G 88 KD                                 24.    G 47, 92 KD       NIL                                                  25.    M 47 KD, G 47 KD  NIL                                                  26.    M 40, 47 KD                                                                   G 40, 47 KD       NIL                                                  27.    M 47 KD, G 47 KD  M 88, 84, 51, 40 KD,                                                          G 88, 84, 51, 40 KD                                  28.    NIL               NIL*                                                 ______________________________________                                         *control serum, so that 6 SLE sera were in the experimental group (see        results).                                                                

IX. C.guilliermondii meningitis/septicaemia

    ______________________________________                                               C. albicans      A. fumigatus                                          ______________________________________                                        29.      M 40 KD, G 40 KD   NIL                                                        M 47 KD, G 47 KD (trace)                                             ______________________________________                                    

RESULTS EPITOPE MAPPING OF HUMAN HSP 90

    ______________________________________                                              Human                                                                         HSP 90                                                                  Epitope                                                                             position   Sequence  Recognised by:-                                    ______________________________________                                        1.    57         KIRY      1/3 invasive aspergillosis                                                    1/1 aspergilloma                                                              4/6 SLE sera                                       2.    101        NNLGTI    7/9 disseminated candidiasis                                                  47 KDa + (including                                                           seroconversion patient*)                                                      1/1 malaria                                                                   1/1 C. quilliermondii                                                         meningitis                                         3.    210        QFIGYPI   2/9 disseminated                                                              candidiasis 47 kDa +                                                          including *seroconverted                                                      patient                                                                       3/3 invasive aspergillosis                                                    (survivors)                                                                   1/2 allergic broncho-                                                         pulmonary aspergillosis                                                       1/6 SLE sera                                       4.    271        KKIK      5/9 disseminated                                                              candidiasis 47 kDa +                                                          including *seroconverted                                                      patient                                                                       2/3 invasive aspergillosis                                                    (survivors)                                                                   3/3 invasive aspergillosis                                                    (fatal)                                                                       1/1 aspergilloma                                                              1/4 SLE sera                                                                  1/1 S. faecalis endocarditis                       5.    404        KILKVIRK  5/9 disseminated                                                              candidiasis including                                      (100% con-     *seroconversion patient                                        served candida)                                                                              3/3 invasive aspergillosis                                                        (survivors)                                                                   1/1 invasive aspergillosis                                                    (fatal, case no 12)                                                           1/1 malaria                                        6.    497        SKEQV     2/9 disseminated candidiasis                               (Candida       3/3 invasive aspergillosis                                     SIKAV)         (survivors)                                                                       1/3 invasive aspergillosis                                                    (fatal)                                                                       1/1 aspergilloma                                                              3/6 SLE                                                                       1/1 malaria                                        7.    549        GLELPE    2/9 disseminated                                                              candidiasis including                                      (Candida       *seroconversion patient                                        FELEES)        3/4 invasive aspergillosis                                                        (survivors)                                                                   1/2 allergic                                                                  bronchopulmonary                                                              aspergillosis                                                                 1/1 aspergilloma                                                              4/6 SLE                                                                       1/1 JK endorcarditis                               8.    580        LDKK      5/9 disseminated candidiasis                               (Candida       1/1 invasive aspergillosis                                     LGDO)          4/6 SLE                                                                           1/1 malaria                                                                   1/1 S. faecalis endocarditis                       9.    607        WTAN      2/9 disseminated candidiasis                                                  including *seroconversion                                                     patient                                                    (Candida       3/3 invasive aspergillosis                                     WSAN)          (survivors)                                                                       1/1 aspergilloma                                                              1/1 malaria                                                                   1/1 S. faecalis endocarditis                       10.   625        NSTMGY    5/9 disseminated                                                              candidiasis including                                                         *seroconversion patient                                    (Candida       3/3 invasive aspergillosis                                     TTMSSY)        (survivors)                                                                       1/3 invasive aspergillosis                                                    (fatal)                                                                       1/1 malaria                                                                   1/1 S. faecalis endocarditis                       11.   642        PIVET     2/3 invasive aspergillosis                                 (Candida       2/2 allergic                                                   PIIKE)         bronchopulmonary                                                                  aspergillosis                                                                 3/6 SLE                                            12.   655        KNDK      4/9 disseminated candidiasis                               (Candida       including *seroconversion                                      (AEDK)         patient                                                                           3/3 invasive aspergillosis                                                    (survivors)                                                                   2/3 invasive aspergillosis                                                    (fatal)                                                                       2/2 allergic                                                                  bronchopulmonary                                                              aspergillosis                                                                 1/1 aspergilloma                                                              5/6 SLE                                            ______________________________________                                         *seroconversion patient--refers to patient 6 whose initial serum was          antibody negative, but, on subsequent recovery from the disease, tested       antibody positive.                                                       

Conclusion

Epitopes 1,3,4,6,7,8,11 and 12 produced a positive response with serafrom SLE patients, i.e. the epitope regognised an autoimmune antibody,and may be classed as autoantibody domains.

Epitopes 2,5,9 and 10 appear to be fungal specific, with epitope 2 beinga potential candidal epitope and epitope 9 a potential aspergillosisspecific epitope. In addition, epitopes 2,5,9 and 10 may be candidatesfor potential malarial epitopes, and epitopes 9 and 10, potentialS.faecalis epitopes.

EXAMPLE 2

The peptide LKVIRKNIVKKMIE cys was used to raise the monoclonal seraused in GB 2 034 504. However, the detailed immunogenicity of thispeptide is unknown. It was decided to investigate this by replacing eachof the amino acids in the above peptide to see the effect, if any, onimmunogenicity.

The necessary peptides were first synthesised using conventionaltechniques and immunogenicity measured using the human sera containingthe monoclonal antibody described above. It was also decided to examinea wider epitope, namely NKILKVIRKNIV.

The protocols and antibody concentrations were the same as thosedescribed above in Example 1.

Peptides evaluated

    ______________________________________                                        Peptide 1    LKVIRKNIV                                                                     evaluated for changes in LKVIRK                                  Peptide 2    NKILKVIRK                                                                     evaluated for changes in KILK                                    Peptide 3    LKVIRKNIV                                                                     evaluated for changes in KNIV                                    ______________________________________                                    

So that in the total sequence the

    ______________________________________                                         ##STR1##                                                                 

    ______________________________________                                        Peptide                                                                             2        1         3      changes were examined                         ______________________________________                                    

Sera assayed

    ______________________________________                                        1.       Monoclonal specfifc to LKVIRKNIVKKMIE - cys                                   against peptides 1 and 3.                                            2.       Human sera.                                                          ______________________________________                                    

    ______________________________________                                        Patient                                                                       Number    Sera      Diagnosis                                                 ______________________________________                                        2         single    Post oesophagectomy, blood culture                                            positive C. albicans.                                     6         paired.sup.a                                                                            Post cholecystectomy, blood                                                   culture positive C. albicans in two                                           sets of blood culture.                                    30        paired.sup.a                                                                            Neutropenia, leukaemia, blood                                                 culture positive C. tropicalis in                                             three sets of blood culture.                              31        paired.sup.a                                                                            Neutropenia, leukaemia, blood                                                 culture positive C. parapsilosis in                                           two sets of blood culture.                                16        paired.sup.a                                                                            Survivor, invasive aspergiliosis                                              acute leukaemia.                                          33        single    P. vivax malaria                                          34        single    P. falciparum malaria                                     35        single    P. falciparum malaria                                     20        single    P.vivax malaria                                           24        single    SLE                                                       ______________________________________                                         .sup.a paired means that there is an early serum which was antibody           negative which is compared to an antibody positive second serum.         

ANTIBODY PROFILES

    ______________________________________                                        Patient No. C. albicans   A. fumigatus                                        ______________________________________                                        2           M 47 KD,      M/G 88,84 KD (trace)                                            G 47 KD       combined                                            6 Post serum                                                                              M 40, 47 KD,                                                                  G 40, 47 KD   NIL                                                 30 Post serum                                                                             M 47 KD,                                                                      G 47 KD       NIL                                                 31 Post serum                                                                             M 47 KD,                                                                      G 47 KD       G 40 KD                                             16          NIL           M 88, 84 KD,                                                                  G 88, 84 KD                                         33          M 47 KD,                                                                      G 47 KD       NIL                                                 34          M 92, 47 KD,                                                                  G 47 KD       G 88 KD                                             35          M 47 KD       NIL                                                 20          M 92 KD,                                                                      G 47 KD       G 51, 40 KD                                         24          G 47 KD       NIL                                                 ______________________________________                                    

Results:

1. Monoclonal specific to LKVIRKNIVKKMIE--cys

Peptide 1--LKVIRKNIV

ELISA values measured at 30 minutes, antibody dilution 1:200

Control LKVIRKNIV average ELISA OD=0.923

(controls)

Substituted amino acid

    ______________________________________                                        (resultant OD)                                                                L         K       V        I     R      K                                     ______________________________________                                        A    0.875    1.102   0.594  0.526 0.111  0.132                               C    0.849    0.797   0.527  0.324 0.125  0.119                               D    0.909    1.208   0.730  0.195 0.126  0.138                               E    1.005    1.183   1.021  0.977 0.141  0.135                               F    1.052    0.760   0.512  0.483 0.116  0.108                               G    0.054    1.016   0.525  0.161 0.131  0.133                               H    0.798    0.926   0.905  0.454 0.107  0.190                               I    1.021    0.714   0.491  control                                                                             0.117  0.12                                K    0.392    control 0.881  0.486 0.092  control                             L    control  0.946   0.673  0.659 0.122  0.114                               M    0.836    1.075   0.508  0.715 0.118  0.364                               N    0.764    1.223   0.769  0.306 0.124  0.137                               P    0.77     0.662   0.612  0.489 0.095  0.132                               Q    0.656    1.073   0.813  0.602 0.120  0.231                               R    0.668    1.032   0.657  0.538 control                                                                              0.591                               S    0.500    1.026   0.620  0.434 0.121  0.146                               T    0.693    0.944   0.740  0.760 0.127  0.139                               V    0.788    0.799   control                                                                              0.977 0.126  0.124                               W    1.022    0.991   1.082  0.895 0.104  0.115                               Y    0.584    1.007   1.196  0.788 0.110  0.119                                    (L)      (K)     (V)    (I)   (R)    (K)                                 ______________________________________                                    

These results show the importance of the amino acids IRK, with RK beingirreplaceable, antibody binding being negligible upon subsitution ofthese amino acids.

Peptide 3--LKVIRKNIV

ELISA values measured at 30 minutes, antibody dilution 1:200

Control LKVIRKNIV average ELISA OD=0.842

    ______________________________________                                               K     N           I       V                                            ______________________________________                                        A        0.101   0.615       0.109 0.134                                      C        0.105   0.196       0.122 0.148                                      D        0.100   0.344       0.100 0.120                                      G        0.102   0.446       0.101 0.113                                      F        0.101   0.166       0.142 0.456                                      G        0.099   0.331       0.100 0.124                                      H        0.107   0.655       0.114 0.123                                      I        0.100   0.169       control                                                                             0.938                                      K        control 0.746       0.095 0.136                                      L        0.103   0.248       0.268 0.831                                      M        0.099   0.630       0.283 0.430                                      N        0.101   control     0.103 0.107                                      P        0.101   0.247       0.107 0.095                                      Q        0.103   0.596       0.103 0.112                                      R        0.192   0.819       0.105 0.121                                      S        0.104   0.942       0.127 0.128                                      T        0.101   0.691       0.181 0.227                                      V        0.100   0.210       0.765 control                                    W        0.098   0.139       0.106 0.123                                      Y        0.102   0.145       0.107 0.105                                      ______________________________________                                    

These results show the importance of amino acids KNIV.

The total epitope is I R K N I V with the underlined amino acidsvirtually impossible to replace.

2. Human Sera

All ELISA performed combined IgM and IgG at 30 minutes. Antibodydilution 1:200

Peptide 1

    ______________________________________                                                         Number of amino acids                                                         giving a reduction to a                                                       maximum of 70% control                                       Patient Number                                                                         Average ELISA L     K    V   I   R   K                               ______________________________________                                        2        1.01          14    4    14  16  5   8                               6 Pre    0.1           not applicable                                         Post     0.501         16    9    16  15  4   3                               30 Pre   0.22          not applicable                                         Post     0.752         9     0    11  15  2   1                               31 Pre   0.1           not appiicabie                                         Post     0.648         14    0    15  17  4   0                               16 Pre   0.347         not applicable                                         Post     0.647         10    2    17  12  2   0                               33       1.18          6     1    12  15  2   0                               34.sup.a 0.6           12    0    6   6   0   0                               35       1.24          8     2    7   7   2   2                               20       1.3           10    1    8   8   1   1                               24       0.379         not applicable                                         ______________________________________                                         .sup.a serum examined at 1:1000 dilution. where ELISA readings were below     0.4, these results were deemed not applicable as they were around the         background level.                                                        

Peptide 2

    ______________________________________                                                         Number of amino acids                                                         giving a reduction to a                                                       maximum of 70% control                                       Patient Number                                                                         Average ELISA   K     I     L   K                                    ______________________________________                                        2          1.37          8     19    19  8                                    6 Pre      0.10          not applicable                                       Post       0.33          not applicable                                       30 Pre     0.22          not applicable                                       Post       1.62          10    18    17  1                                    31 Pre     0.11          not applicable                                       Post       0.58          10    18    17  3                                    16 Pre     0.3115        not applicable                                       Post       0.531         2     17  14    13                                   33                       7     16  12    2                                    34.sup.a   0.47          4     17  16    2                                    35         1.36          10    18  17    3                                    20         1.2           8     16  16    2                                    24         0.32          not applicable                                       ______________________________________                                         .sup.a serum examined at 1:1000 ELISA values less than 0.4 taken as           background, therefore deemed not applicable.                             

Peptide 3

    ______________________________________                                                         Number of amino acids                                                         giving a reduction to a                                                       maximum of 70% control                                       Patient Number                                                                         Average ELISA K     N    I   V                                       ______________________________________                                        2        0.89          0     0    0   0                                       6 Pre    0.01          not applicable                                         Post     0.438         0     0    0   0                                       30 Pre   0.2           not applicable                                         Post     0.52          0     0    3   3                                       31 Pre   0.35          not applicable                                         Post     0.66          0     1    9   4                                       16 Pre   0.3           not applicable                                         Post     0.59          3     8    15  11                                      33       1.03          1     13   11  7                                       34.sup.a 0.566         3     15   14  12                                      35       1.437         0     8    10  3                                       20       1.18          8     14   15  15                                      24       0.37          not applicable                                         ______________________________________                                         .sup.a serum examined at 1:1000 ELISA values less than 0.4 taken as           background, therefore deemed not applicable.                             

EXAMPLE 3

A library of immunoglobulin heavy and light chain variable (V) genes wasprepared from the peripheral blood lymphocytes of a patient who hadrecovered from systemic candidiasis. The library was enriched andscreened for activity to LKVIRKNIVKKMIE and NKILKVIRKNIVKK and theresulting human recombinant antibodies examined for therapeutic activityin a mouse model of systemic candidiasis.

Experimental Details

The library was produced essentially as described by Marks et al (J MolBiol 1991; 222: 581-597) using the pCANTAB 5 vector, which is nowcommercially available as part of a kit from Pharmacia (Milton Keynes,UK). The heavy and light chain V genes, obtained from cDNA prepared fromthe mRNA of peripheral blood lymphocytes of a patient recovering fromsystemic candidiasis, were randomly combined and subcloned into NotI/Sfi I digested pCANTAB 5. The resulting single chain Fv fragments(ScFv), expressed on the surface of phage, were enriched by panningagainst specific synthetic peptide epitopes, including LKVIRKNIVKKMIEand NKILKVIRKNIVKK. Then immunoblotting (see Example 4) was used toidentify individual clones with activity against the 47 kd antigen ofCandida albicans. Two strongly positive recombinant antibodies wereselected for further study: A2 (panned against LKVIRKNIVKKMIE) and B3(panned against NKILKVIRKNIVKK).

Female Balb/c mice were injected intravenously with a lethal dose ofCandida albicans. Two different age groups were examined - mature miceweighing around 20 g (experiments 1 and 2) and young mice weighingaround 12 g. Two different batches of human recombinant B were examined(experiment No. 1 against No's 2 and 3). In experiment 1 thisrecombinant antibody was compared against human recombinant antibody Aand helper phage M13 K07, and 200 μl of saline, M13 K07 or humanrecombinant antibody was given approximately 2 hours after iv challengewith Candida. In experiments 2 and 3, 100 ul of saline or B3 was given 1hours after iv challenge with Candida.

Results

    ______________________________________                                                       Av.Wt.                                                                        of mice  Treatment Group                                                                         % mortality at 24 h                         Expt. No.                                                                            CFU     (g)      (No. of mice)                                                                           (No. dead)                                  ______________________________________                                        1      10.sup.8                                                                              21 ± 3 g                                                                            Saline (11)                                                                             73% (8)                                            10.sup.8                                                                              21 ± 3 g                                                                            M13K07 (17)                                                                              76% (13)                                          10.sup.8                                                                              21 ± 3 g                                                                            A (16)     94% (15)                                          10.sup.8                                                                              21 ± 3 g                                                                            B (10)    10% (1)                                     2      10.sup.7                                                                              19 ± 2 g                                                                            Saline (10)                                                                             40% (4)                                            10.sup.7                                                                              19 ± 2 g                                                                            B (10)    10% (1)                                     3      10.sup.8                                                                              12 ± 2 g                                                                            Saline (16)                                                                             25% (4)                                            10.sup.7                                                                              19 ± 2 g                                                                            B (16)     0% (0)                                     ______________________________________                                    

Comment

Younger mice appear to be relatively resistant to Candida albicans (ananalogous situation occurs in humans). Human recombinant antibody Brepeatedly showed therapeutic activity against Candida--unlike humanrecombinant antibody A.

EXAMPLE 4

Human recombinant antibodies B3 and A2 were examined forcross-reactivity against Streptococcus faecalis and Corvnebacteriumjeikeium.

Experimental details

Immunoblots of Candida albicans, Corvnebacterium jeikeium andStreptococcus faecalis were prepared as previously described (Matthews,Epidemiol. Infect. 1991; 107: 273-283; Burnie et al, J. Clin. Path.1987; 40: 1149-1158). Free-protein binding sites were blocked withbovine serum albumin (BSA), prior to incubating each blot with the phagesuspension (B3 or A2) for 2 hours at room temperature, with gentleagitation. They were then washed five times over 30 min in 0.05% Tween20-Saline, and incubated for 90 minutes with anti-M13 antibody(commercially available from 5'-3', Inc. Boulder, USA), diluted 1:1000in 3% BSA. Washing was repeated and then they were incubated withalkaline-phosphase conjugated anti-goat antibody (diluted 1:1000), fromSigma, for 1 hours. Washing was repeated and then the blots weredeveloped with the colour substrate BCIP NBT as previously described(Matthews 1991).

Results

B3 and, to a less extent, A2, cross-reacted with Streptococcus faecalisand Corvnebacterium jeikeium as well as Candida albicans. Several bandswere produced. In the case of S.faecalis these were at about 112 kd,88-90 kd and 32 kd. In the case of C.jeikeium bands occurred at about160 kd, 52 kd, 47 kd and 40 kd. In the case of C.albicans a Prominentband occurred at 47 kd.

Comments

Human recombinant antibodies B3 and A2 cross-reacted with S.faecalis andC.jeikeium and may have therapeutic potential against these and otherGrampositive bacteria.

EXAMPLE 5

Dot--Immunobinding Assay

Material and Methods

1 μl of human serum was dotted onto a sheet of nitrocellulose membrane(Biorad Laboratories, California). This was allowed to dry for 10minutes. It was blocked for 1 hour at 37° C. with 3% Bovine SerumAlbumen in Tris-buffered saline pH 7.5. After being washed in0.9%/weight/volume/saline--0.05% Tween 80 five times over 30 minutes itwas incubated for 1 hour at room temperature with the human recombinantantibody B3 diluted 1 in 5. It was then washed for 30 minutes, asdescribed above, and further incubated with the anti-M13 horseradishperoxidase conjugate (Pharmacia Ltd), diluted 1 in 5,000 in 3% bovineserum albumin in Tris-buffered saline. After a further wash, this wasdeveloped with the 3-amino-9-ethylcarbazole substrate (0.67 ml of asolution of 0.4 gram 3-amino-9-ethylcarbazole/100 ml dimethyl formamidein 10 mls of 0.1M sodium acetate (pH 5.2)). This was activated with 10μl of 30% H₂ O₂.H₂ O₂. The blot was incubated for 30 minutes and washed.The results were compared with a positive control of 1 μl of candidalpressate (100 mg/ml) and graded as negative (nil), weakly positve(trace) and positive (equivalent to the result from the candidalextract).

Dot Immunobinding Assay with Recombinant Antibodies

Results

Control sera (sera from patients with no evidence of disseminated ororal candida, either with leukaemia or after major gastrointestinalsurgery).

    ______________________________________                                        Titre:     NIL          Trace  Positive                                       ______________________________________                                        No. of Sera:                                                                             79           2      NIL                                            ______________________________________                                    

The two trace positive patients came from a group of five with anunderlieing septicaemia due to a Corynebacterium jeikeium.

Colonized Patients (sera from patients with a colonized site eitheroral/wound/vaginal/intraveneous line/urine).

    ______________________________________                                        Titre      NIL          Trace  Positive                                       ______________________________________                                        Oral       6            1                                                     Wound      1                                                                  Vaginal    1                                                                  Intravenous                                                                              3.sup.a                                                            Urine      2                   1.sup.b                                        ______________________________________                                         .sup.a includes 1 case of C.parapsiliosis colonization                        .sup.b required treatment Amphotericin B.                                

Systemic patients

Defined as either (1) two sets of positive blood cultures taken at least24 hours apart from two different intravenous sites or (2) cultural andhistopathological evidence obtained at postmortem.

Each patient is reported as the maximum antigen titre detected if aseries of samples was available.

    ______________________________________                                                    Maximum Antigen Titre Detected                                    Causative yeast                                                                             Nil        Trace    Positive                                    ______________________________________                                        Candida tropicalis                                                                          1          1                                                    Torulopsis glabrata               1                                           Candida parapsilosis              1                                           Candida albicans                                                                            2          3        17                                          ______________________________________                                    

Comments

Antigen detected by dot immunobinding with the B3 recombinant antibodydistinguished control sera and sera from colonized patients from thesera of patients with systemic candidiasis.

CONCLUSIONS

Concerning heat shock protein epitopes

The amino acids underlined below are vital, i.e. irreplaceable, to thefunction of the epitope as an immunogen. In addition, sera of patientswith different diseases produce antibodies which recognise slightlydifferent amino acids within the same epitope.

Case 24, an SLE patient acted as a positive control, as, although thispatients sera was positive for the candidal 47 KD protein, it did notrecognise any of the 3 peptides of the epitope of the present invention.

    ______________________________________                                        Monoclonal epitope      L K V I R K N I V                                     C. albicans epitope                                                                         case 2    N K I L K V I R K N I V                                             case 6    N K I L K V I R K N I V                               C. tropicalis case 30   N K I L K V I R K N I V                               C. parapsilosis                                                                             case 31   N K I L K V I R K N I V                               A. fumigatus  case 16   N K I L K V I R K N I V                               Malaria:. vivax                                                                             case 33   N K I L K V I R K N I V                                             case 20   N K I L K V I R K N I V                               falciparum    case 34   N K I L K V I R K N I V                                             case 35   N K I L K V I R K N I V                               ______________________________________                                    

The monoclonal described in GB 2 034 504 reacts with an epitope whichonly represents part of the epitope for yeast infection(C.albicans/C.tropicalis/ C.parapsilosis). Infections due toC.tropicalis, A.fumigatus and malaria are more immunoreactive to theKILK epitope. Infections due to malaria and A.fumigatus also recognisean epitope containing NIV.

Thus a more active antibody recognising stress proteins produced in awider range of diseases than that described in GB 2 034 504 may beproduced against the epitope N K I L K V I R K N I V (i.e. antibody B3).

Concerning antibodies against heat shock protein epitopes

The data demonstrate that antibodies raised against X X X L X V I R K XI V and/or X X I L X V I X X X X X, where X is any amino acid,particularly human recombinant antibodies, show protection in the mousemodel of systemic candidiasis.

It will be appreciated that it is not intended to limit the invention tothe above examples only, many variations, such as might readily occur toone skilled in the art, being possible, without departing from the scopethereof as defined by the appended claims.

We claim:
 1. An isolated epitope of up to nine amino acids from humanHSP90 which cross-reacts with an antibody against at least one otherHSP90 protein, said epitope comprising the amino acid sequence Lys IleArg Tyr or Asn Asn Leu Gly Thr Ile.
 2. An epitope according to claim 1,wherein the HSP90 protein comprises a malarial stress protein.
 3. Anepitope according to claim 1, wherein the HSP90 protein comprises afungal stress protein.
 4. An epitope according to claim 1, wherein theHSP90 protein comprises a bacterial stress protein.
 5. An epitopeaccording to claim 3, wherein the fungal stress protein comprises aprotein selected from the group consisting of a Candidal 92 and 47 KDaproteins.
 6. An epitope according to claim 3, wherein the fungal stressprotein comprises a protein selected from the group consisting ofAspergillus 40, 51 and 88/84 proteins.
 7. An epitope according to claim3, wherein the fungal stress protein comprises a Pneumocystis cariniiprotein.
 8. An epitope according to claim 4, wherein the bacterialstress protein comprises a Streptococci 90 KD stress protein.
 9. Anepitope according to claim 4, wherein the bacterial stress proteincomprises a Corynebacterial 86 KD or Corynebacterial 52 KD orCorynebacterial 86 and 52 KD stress protein.
 10. A diagnostic kitcomprising an epitope according to claim 1 and instructions forperforming a diagnostic test.
 11. A diagnostic kit according to claim10, wherein the diagnostic test is a dot immunosorbent assay, aradioimmunoassay or a latex agglutination assay.
 12. An isolated epitopeof up to nine amino acids from human HSP90 which cross-reacts with anantibody raised against at least one of the HSP90 proteins of Candida,Streptococcus faecalis, Corynebacterium jeikeium, Aspergillus fumigatus,Plasmodium vivax and Plasmodium falciparum, wherein said epitopecomprises the amino acid sequence Lys Ile Arg Tyr or Asn Asn Leu Gly ThrIle.